BCL6 inhibitors as anticancer agents

ABSTRACT

The invention provides compositions and methods for blocking the BCL6 BTB domain with small molecule, non-peptide compounds as disclosed and claimed herein. BCL6 is a transcriptional repressor of the BTB-POZ (brie a brae, tramtrack, broad complex/pox virus zincfinger) family of proteins. It is required for normal development of germinal center (GC) B-cells and is also the most commonly involvedoncogene in diffuse large B-celllymphomas (DLBCLs), and constitutive expression of BCL6 in GC B-cells causes DLBCL in mice.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage filing under 35 U.S.C. 371from International Application No. PCT/US2014/042556, filed Jun. 16,2014, and published as WO 2014/204859 and published on Dec. 24, 2014,which claims the priority of U.S. Ser. Nos. 61/836,043, filed Jun. 17,2013; 61/845,255, filed Jul. 11, 2013; and 61/939,827, filed Feb. 14,2014; the disclosures of which are incorporated by reference in theirentireties.

BACKGROUND

BCL6 is a transcriptional repressor of the BTB-POZ (bric à brac,tramtrack, broad complex/pox virus zinc finger) family of proteins. Itis required for normal development of germinal center (GC) B-cells andis also the most commonly involved oncogene in diffuse large B-celllymphomas (DLBCLs), and constitutive expression of BCL6 in GC B-cellscauses DLBCL in mice. DLBCLs are aggressive tumors that arise fromgerminal center (GC) B-cells and are the most common form ofnon-Hodgkin's lymphomas. BCL6 is required for survival of DLBCL cellsand can limit their ability to respond to DNA damaging agents. It isalso frequently expressed in follicular lymphomas (FLs), and may berequired for survival of these tumors as well. DLBCL and FL collectivelyconstitute ˜60-70% of B-cell lymphomas and the incidence of these tumorshas been rising in recent decades. BLC6 binds to the SMRT co-repressorthrough a tight and unique interaction mediated by the N-terminalBTB/POZ domain of BCL6. Peptides that mimic the SMRT interface candisplace SMRT from BCL6, de-repress BCL6 target genes and kill DLBCLcells. BCL6 has an N-terminal BTB domain that mediates transcriptionalrepression and a C-terminal C2H2 zinc finger that binds to a specificDNA consensus sequence. The two regions are linked by a secondrepression domain (RD2) that also has repressor activity. The BTB domainof BCL6 recruits the SMRT, N-CoR, and BCoR corepressors. The minimalbinding domain of the SMRT, N-CoR, and BCoR corepressors maps to aconserved 17 amino acid sequence (BBD, BCL6 Binding Domain) that bindsto a “lateral groove” motif on the BCL6 BTB domain dimer. This lateralgroove/BBD interaction is required for the BCL6 domain to recruit theSMRT, N-CoR, and BCoR corepressors, and is essential for the repressionactivity of BCL6. The peptides contain a common aromatic residue thatfits into a pocket within the lateral groove and they adopt a similarpseudo-structure upon binding. Also, the SMRT, N-CoR, and BCoR BBDs bindspecifically to the BCL6-BTB but not to other BTB domains from any otherprotein member of the family.

Approximately 80% of patients with DLBCL responded to conventionalchemotherapy consisting of cyclophosphamide, doxorubicin, vincristine,and prednisone (CHOP), but less than 40% were likely to be cured.Randomized trials showed that the addition of rituximab (R) toconventional CHOP chemotherapy improves outcomes including overallsurvival, establishing R-CHOP as the standard of care. A recent analysisof outcomes on the US Intergroup Trial (E4494) comparing CHOP to R-CHOPand maintenance rituximab indicated that the benefit observed with theaddition of rituximab was attributable to a powerful effect on the BCL6negative cases only. Cases positive for BCL6 did not benefit from theaddition of rituximab to CHOP chemotherapy. The mechanism by which theaddition of rituximab to CHOP improves outcomes selectively in the BCL6negative cases is unknown but could be related to antibody-dependentcellular cytotoxicity, complement-mediated cytotoxicity, induction ofapoptosis or an as yet uncharacterized effect unique to BCL6 negativeDLBCL. These findings underscore the fact that the DLBCLs represent atleast two biologically distinct diseases that will require differenttreatment approaches to improve upon current outcomes. To date, clinicaltrials for DLBCL have not distinguished between the different subgroups.Therapeutic strategies must be designed to specifically target BCL6positive and negative DLBCL based upon their unique biologicaldifferences.

SUMMARY

The invention provides, in various embodiments, a method of disruptingBCL6 BTB domain interactions with corepressors, in B-cells, comprisingexposing the B cells to an effective concentration of a compound thatblocks the lateral groove of BCL6; a method of inhibiting DLBCL tumorgrowth, or causing DLBCL tumor regression, or both, in a mammal,comprising administering to the mammal an effective dose of a compoundthat blocks the BTB lateral groove of BCL6; a method of inhibitingtranscriptional repression induced by a complex of BCL6 with SMRT orother corepressor proteins in cancer cells, comprising exposing thecancer cells to an effective concentration of a compound that blocks theBTB lateral groove of BCL6; and a method of treatment of a patientafflicted with cancer, comprising administering to the patient aneffective dose of a compound that blocks the BTB lateral groove of BCL6.The practice of these methods can be accomplished using BCL6-bindingcompounds of the invention as disclosed herein.

The invention further provides compounds of various formulas, effectivefor carrying out a method of the invention, as disclosed and claimedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of bioactivities of compounds of the 1085 series ina reporter assay in which a GAL4-DNA binding domain (DBD)-BCL^(BTB)fusion construct was co-transfected with a luciferase reporter plasmidcontaining GAL4 DBD binding sites, (GAL4)5TK-Luc in 293T cells. The foldchange in the repressor activity of the BCL6^(BTB) domain, shown ingraphic form, was determined in the presence of either the compounds atdifferent concentrations, or DMSO alone (2 μL/mL), used as a vehicle ofdilution of the compounds, and was controlled for nonspecific effects ontranscription by normalizing to the activity of the GAL4-DBD alone. Theresults presented are the average and standard deviation of threeindependent experiments (**=p<0.005; *=p<0.05 t-test).

FIG. 2 shows a graph of the compounds of the 1085 series in a reporterassay performed under the same conditions as in the assay of FIG. 1, buttransfecting cells either with GAL4 DNA binding domain (DBD)-HIC1-BTB,PLZF-BTB, or Kaiso-BTB fusion constructs instead of BCL6-BTB.

FIG. 3 shows a graph of bioactivities of compounds of the 2099 seriesanalogous to the results shown for FIG. 1.

FIG. 4 shows a graph of bioactivities of compounds of the 2071 seriesanalogous to the results shown for FIG. 1.

FIGS. 5, 6A and 6B show graphs of bioactivities of compounds of the 3033series analogous to the results shown for FIG. 1.

FIGS. 7A and 7B shows graphs of bioactivities of compounds of the 3033series and other compounds analogous to the results shown for FIG. 1.

FIGS. 8A and 8B depicts structures and biodata for the 4044 series incomparison with compound 1020.

FIGS. 9A and 9B shows biodata for compounds of the 3077 irreversibleinhibitor series.

FIG. 10 shows structural and computational data related to compounds ofScaffold 2.

FIGS. 11A, 11B and 11C depicts structures and biodata for additionalcompounds of Scaffold 2.

FIG. 12 shows structural and computational data for Scaffold 3.

FIG. 13 shows structural and computational data for Scaffold 4.

FIG. 14 shows structural and computational data for Scaffold 5.

FIG. 15 shows structural and computational data for Scaffold 6.

FIG. 16 shows structural and computational data for Scaffold 7.

FIG. 17 shows structural and computational data for Scaffold 8.

FIG. 18 shows structural and computational data for Scaffold 9.

FIG. 19 shows biodata for a compound of Scaffold 9.

FIG. 20 shows structural and computational data for Scaffold 10.

FIG. 21 shows structural and computational data for Scaffold 11.

FIG. 22 shows biodata for a compound of Scaffold 11.

FIGS. 23A-D shows structures of exemplary compounds of Scaffold 12.

FIGS. 24-27 show biodata for compounds of Scaffold 12, analogous to theresults shown for FIG. 1.

DETAILED DESCRIPTION

BCL6 was first identified as the most frequently deregulated gene indiffuse large B-cell lymphomas (DLBCL). The translocations of this genecause BCL6 to be constitutively expressed downstream of heterologouspromoter regions. DLBCLs are aggressive tumors that arise from germinalcenter (GC) B-cells and are the most common form of non-Hodgkin'slymphomas. GCs are transient structures that form within lymphoidfollicles triggered by exposure to antigenic stimulation. The currentstandard treatment of DLBCL involves the RCHOP regimen. Thispolychemotherapy regimen can cure approximately 80% of GCB-type DLBCLsand 40% of ABC-DLBCLs. Hence many DLBCL patients still relapse fromchemotherapy and need better treatments. BCL6 inhibitors are effectivein both ABC and GCB type DLBCLs, and are highly active even in lymphomacells from chemo-resistant patients. Moreover BCL6 inhibitors synergizewith chemotherapy and could be used to help eradicate DLBCLs that wouldotherwise relapse. Given how powerful and yet non-toxic BCL6 targetedtherapy appears to be, it seems that BCL6 inhibitors could serve as ananchor for combination therapies without fear of significantcross-toxicities. Also, BCL6 inhibitors are active in two of the worstsubtypes of Acute B-Lymphoblastic leukemia (B-ALL), those with MLLtranslocations and those with BCR-ABL translocations. Most of thesepatients die of their disease. There is a clear need for a meaningfultherapeutic agent that could help these patients.

Likewise, the therapy of acute myeloid leukemia (AML) has changed littleover the past 30 years and most patients still die of their disease.Again BCL6 therapy may provide an important new modality for thesepatients. Targeting BCL6 requires disrupting protein-proteininteractions and our BCL6 inhibitors approach is one of the first orpossibly the first example where this has been achieved for therapeuticeffect against an oncogenic transcription factor. The only known BCL6inhibitors that target the lateral groove are the BPI (BCL6 peptideinhibitor) peptides and 79-6, described below. However, BPI suffers fromlack of oral bioavailability, potential immunogenicity, and high costwhile 79-6 is limited with respect to affinity and stability. This studyis innovative in two aspects: novel sweet spots on the BTB lateralgroove of BCL6 were identified, including the aromatic, arene, and HDCHpockets, and exploited to facilitate the first truly rational design ofsmall molecule BCL6 inhibitors. Pocket characterization and inhibitordesign were driven by a novel target-based CADD method, SiteIdentification by Ligand Competitive Saturation (SILCS, see Guvench, O.,and MacKerell, A. D., Jr. “Computational Fragment-Based Binding SiteIdentification by Ligand Competitive Saturation, PLoS ComputationalBiology, 5: e1000435, 2009, PMC2700966), which can be used together withmedicinal chemistry, structural studies and biological assays in aniterative approach to optimize the pharmacodynamic (PD) andpharmacokinetic (PK) properties of the inhibitors. Second, a cysteineresidue located in the novel HDCH binding pocket in the lateral groovewas targeted to develop specific, irreversible BCL6 inhibitors. Theproposed research has the potential to treat other important humancancer such as FLs, various forms of leukemia and breast cancer, andteach us how the inhibition of BCL6 and subsequent gene repressionaffects cancer

To identify novel BCL6 inhibitors, we first used applied the SILCSmethodology using the crystal structure of the BCL6-BTB/SMRT-BBD complex(PDB ID 1R2B). The resulting SILCS FragMaps, which represent theaffinity pattern of the protein for different types of functionalgroups, was used to design novel compounds targeting the BTB lateralgroove. Top scoring designed compounds were then synthesized andevaluated using functional assays including microscale thermophoresisprotein binding, NMR, DLBCL differential killing and BCL6 BTB domainreporter assays. We therefore synthesized different groups of compounds.

A retro-inverso peptide that blocks the oncogenic activity of BCL6 wasdeveloped; studies done by certain of the inventors herein have led tothe development of a recombinant peptide containing the SMRT BBD and apTAT protein transduction domain that occupies the BCL6 BTB lateralgroove and prevents binding of the SMRT, N-CoR, and BCoR corepressors.The BPI (BCL6 peptide inhibitor) was specific to BCL6 and shown toactivate BCL6-target genes in DLBCL cells. Intraperitoneal injections ofBPI in mice reproduced the BCL6 null phenotype. BPI also had specificand potent anti-lymphoma activity, inducing apoptosis in a panel of BCL6expressing DLBCL cell lines but no effect for BCL6 negative cell lines.The BPI peptide analog is disclosed in PCT/US2009/003483, published asWO 2010/008436.

These data demonstrate that BCL6 is a therapeutic target that can beeffectively and specifically blocked by occluding its BTB domain lateralgroove. Although the peptide is very stable, it is extremely expensiveand difficult to produce due to the need for D-aminoacid series in apure state. Therapeutic targeting of the BCL6 lateral groove isdisclosed in PCT/US2004/042418, published as WO2005/058939, (andcorresponding U.S. application Ser. No. 10/582,662, now U.S. Pat. No.7,919,578), filed Dec. 16, 2004, the disclosure of which is incorporatedherein by reference in its entirety.

A small molecule inhibitor of BCL6 that kills DLBCL cells in vitro andin vivo was then developed. Compound 79-6 specifically inhibits BCL6 butnot other BTB domains such as HIC1 (hypermethylated in cancer 1), PLZF(promyeloctic zinc finder), and Kaiso.

Compound 79-6, shown above, is disclosed in PCT/US2007/024571, filedNov. 30, 2006, published as WO2008/066887, and in U.S. Ser. No.12/312,800, now U.S. Pat. No. 8,338,464; the disclosures of whichpublications are incorporated herein by reference in their entireties.Compound 79-6 disrupts BCL6 transcriptional complexes and reactivatesBCL6 target genes. Compound 79-6 selectively kills BCR DLBCL sells. Itis not toxic and suppresses human DLBCL xenografts in mice. Compound79-6 selectively kills primary BCL6+ DLBCL cells. The X-raycrystallographic structure of the BCL6^(BTB)/79-6 complex shows onemolecule of compound 79-6 to bind in each of lateral grooves of the BLC6BTB dimer. This compound provided a very good structural lead, but novelcompounds that are more potent, and that are soluble and stable in watersolution are needed in order to improve efficacy in the treatment oflymphomas in humans. Reference is made to publications cited herein, thedisclosures of which are incorporated by reference in their entireties.

The present invention describes, in various embodiments, novelinhibitors of BCL6 repression activity that act through binding to thelateral groove motif of the BLC6 BTB domain thereby preventing itsinteraction with the co-repressor complex, that are effective for use asanti-lymphoma agents. Various classes have compounds have been devised,as disclosed and claimed herein, that can be used for treatment oflymphomas such as BCL6+ Diffuse Large B-Cell lymphomas. We identifiednovel inhibitors with improved potency that were structurally similar tothe previously discovered lead 79-6.

Based on the X-ray structure of 79-6 with BCL6, we designed analogs thatqueried 1) the substituent(s) on the indolin-2-one ring, and 2) thelinker length between the rhodanine and the carboxylic acid tail, withthe goal of identifying a ligand with one acid group, thereby likelyhaving improved PK properties. These efforts identified 79-6 analog1085, which contains a 5-Cl substituent on the indolin-2-one ring and amono carboxylic acid.

To map the functional group requirement of the 79-6 binding pocket todirect ligand design we have applied the SILCS methodology. In the SILCSmethod a collection of small molecules representative of differentclasses of functional groups compete with each other and with water forthe surface of the protein during a series of MD simulations. From thesesimulations 3D probability distribution maps, termed FragMaps, areobtained which identify regions of the protein surface with whichdifferent types of functional groups have favorable interactions withthe protein, information that can direct ligand design. Notably, SILCSallows for 1) qualitative analysis of the protein-binding pocket toallow visualization and subsequent prediction of syntheticallyaccessible modifications of ligands that should improve activity and for2) quantitative estimates of changes in binding affinity associated withthe addition of the predicted functional groups. Evident is the overlapof aromatic and aliphatic FragMaps, which defines the aromatic pocket,with the indolin-2-one ring of 79-6 and of charged acceptor FragMapswith the acid groups, which defines the acid site. These resultsemphasize the importance of these functional groups for the activity of79-6. Notably, the aromatic and aliphatic FragMaps are extended toencompass the Br atom on the 5 position of the indolin-2-one, indicatingthat this position is the most likely position for a substituent asvalidated in compound 1085. The lack of FragMaps coinciding with therhodanine ring indicates that it is acting as a scaffolding element inthe ligand rather than making a significant contribution to binding. TheFragMaps also indicate that only one of the two carboxylate groups isnecessary to form the key charge-charge interaction with the acid siteassociated with the side chain of Arg28, a result that has beensubsequently validated by the activity of FX-1085. With respect tofuture design options, adjacent to the indolin-2-one ring is anaromatic/aliphatic FragMap, defining the arene pocket.

After testing the effect of 25 different derivatives in a reporter assay(FIG. 1), we identified the new compound 1085 as the most potent andselective one (FIG. 2), with better affinity to the BCL6-BTB domaincompared to 79-6 and even to the natural ligand peptide SMRT. Under theassay conditions, 1085 had a Kd of (5±4)μM, whereas 79-6 had a Kd ˜120μM. The superposition of the 15N-1H HSQC spectra for Bcl6 BTB alone andspectra in the presence of 1085 shows a similar pattern to the oneobserved with the 79-6 BCL6-BTB interaction, confirming that the bindingsite is similar for both compounds. The novel compound also showsdifferential gene expression similar to the silencing of the proteininduces the derepression of BCL6 target genes and prevents thecorepressor complex proteins SMRT or BCOR to be bound to BCL6 that isinteracting with the target sequences. BCL6 is also required for normaldevelopment of germinal center (GC) B-cells. Treatment of immunized micefor 10 days with 100 mg/kg 1085 inhibited the germinal center,confirming the effect of 1085 as a BCL6 inhibitor. This compound hasfavorable pharmacokinetics in vitro and in vivo, is very stable insolution and didn't induce any toxic effects in mice after 10 days oftreatment with 125 mg/kg. As expected, 1085 is a very effective andselective inhibitor of BCL6 dependent GCB DLBCLs in vitro and in vivothat induces tumor regression in 95% of the tumors. Noteworthy, BCL6dependent aggressive ABC DLBCLs were also sensitive to the in vitro andin vivo treatment with 1085, and even primary human ABC DLBCL sampleswere sensitive to the treatment with the new BCL6 inhibitor.

Thus, the invention provides, in various embodiments, a compound of the1085 series, exemplified as shown in Table 1, below, based on the leadstructure of 1085. A reporter assay in which a GAL4-DNA binding domain(DBD)-BCL^(BTB) fusion construct was co-transfected with a luciferasereporter plasmid containing GAL4 DBD binding sites, (GAL4)5TK-Luc in293T cells. The fold change in the repressor activity of the BCL6^(BTB)domain, shown in graphic form in FIG. 1, was determined in the presenceof either the compounds at different concentrations, or DMSO alone (2μL/mL), used as a vehicle of dilution of the compounds, and wascontrolled for nonspecific effects on transcription by normalizing tothe activity of the GAL4-DBD alone. The results presented are theaverage and standard deviation of three independent experiments(**=p<0.005; *=p<0.05 t-test).

TABLE 1 Examples of the 1085 series 1085

1117

1093

1165

1095

1167

1097

1169

1113

2001

1115

2003

2021

2005

2023

2035

2025

2037

2027

2039

2031

2041

2033

3021

3039

Most of the compounds of this family were active in the reporter assay,being able to inhibit the repression activity of BCL6 more than 20%. Themost active compound was 1085, inhibiting the repression activity ofBCL6 approximately 50%. In this assay, the compounds are tested by theircapacity to inhibit the repressor activity of BCL6-BTB, but also need topenetrate the cell membrane and interact with the protein in order toproduce the inhibition, so the permeability of the cell is also tested.

To test the selectivity of the compounds a similar reporter assay wasperformed under the same conditions as in the assay of FIG. 1, buttransfecting cells either with GAL4 DNA binding domain (DBD)-HIC1-BTB,PLZF-BTB, or Kaiso-BTB fusion constructs instead of BCL6-BTB. Resultsare shown in FIG. 2, suggesting that none of the tested compoundssignificantly affected the repression activity of the HIC1, PLZF, orKaiso BTB domains.

50,000 cells per well were treated for 48 hours with differentconcentrations of the compounds in 96 well plates, using DMSO as acontrol. Then, viability was determined with Cell Titer Blue™ (Promega)and growth inhibition was calculated as the percentage of viable cellswith respect to cells treated with DMSO alone. GI₅₀ values weredetermined by dose-response curves. Results are presented in Table 2,below.

TABLE 2 Growth inhibition effects of 1085 compounds on DLBCL cells BCL-6independent BCL-6 dependent Karpas Ly3 Ly7 Ly10 SUDH6 422 Toledo Ly1B50Compound GI₅₀ (μM) 1085 >40 23 ± 9 23.5 ± 0.2 14 ± 7 >40 >40 >401093 >40 30 ± 9  21 ± 12 >40 >40 >40 >401095 >40 >40 >40 >40 >40 >40 >40 1097 >40 >40 18 ± 8 >40 >40 >40 >401113 >40 12 ± 7 >40 >40 >40 >40 >40 1115 22 ± 9 24 ± 6 34 ± 3 26 ±7 >40 >40 >40 1117 21 ± 4 36 ± 1 >40 25 ± 4 >40 >40 >40 1165 17 ± 6 >4025 ± 9 >40 >40 >40 >40 1167 >40 22 ± 1 24 ± 2 29 ± 1 >40 >40 >40 1169 29± 5 28 ± 7  20 ± 10 30 ± 3 27 ± 8 >40 >402001 >40 >40 >40 >40 >40 >40 >40 2003  >6  >6  >6  >6  >6 >6 >62005 >40 >40 21 ± 2 25 ± 6 >40 >40 >40 2021 >40 >40 >40 >40 >40 >40 >402023 >40  21 ± 10 >40 21 ± 3 >40 >40 >40 2025 >40 13 ± 1 15 ± 8 14 ± 222 ± 7 >40 >40 2027 14 ± 3  8 ± 1  6 ± 2  7 ± 1 >40 >40 >402031 >40 >40 >40 >40 >40 >40 >40 2033 >40 >40 >40 >40 >40 >40 >402035 >40 >40 >40 >40 >40 >40 >40 2037 >40 >40 >40 >40 >40 >40 >402039 >40 >40 >40 >40 >40 >40 >40 2041 >40 >40 >40 >40 >40 >40 >40

TABLE 3 Compounds of the 2099 series

371

372

373

374

375

376

377

378

379

380

381

382

383

384

2097

2099

2101

3022

The toxicity of the compound 1085, the most active and specific BCL6inhibitor of this series, was tested. The compound was found to have aselective BCL6-dependent DLBCL inhibitor activity with a GI₅₀ between 14and 23 μM. First, the stability of the compound in the administrationvehicle of 30% PEG-300, 5% Tween-80, and 65% dextrose 5%, was evaluatedat room temperature over a period of up to 8 days by NMR spectroscopy.

No significant changes in the structure were observed after the 8 dayevaluation. The toxic effects of 1085 on mice were then examined. FiveC57BL/6 mice were exposed to daily intraperitoneal (IP) administrationof increasing doses of the compound ranging from 50 to 150 mg/kg overthe course of 7 days, to a cumulative dose of 750 mg/kg, and anotherfive mice were exposed to vehicle only. No toxic effects or otherindicators of sickness, including significant weight loss or tissuedamage (macroscopic or microscopic) were noted. Brain, heart, lung,liver, kidney, bowel, spleen, and bone marrow tissues were examined.Complete peripheral blood counts, biochemistry and liver function testswere normal.

A series of N-substituted oxindoles and oxindole analogs was thenprepared and tested in the same bioassays. Table 3, above, showsexamples of compounds in the series designated the 2099 series.

FIG. 3 shows biodata results indicating the % repression at a 50 μMlevel of the compound relative to control. Tables 4 and 5 showquantitative results of the bioassays on BCL6-dependent andBCL6-independent cell lines.

TABLE 4 Bcl6 dependent Bcl6 independent GI 50 (μM) Ly3 Ly1 SUDHL6 Ly7Toledo K422 WSUDLCL2 Ly1B50 374 >125 >125 48 ± 17 >125 >125 >125 99 ±20 >125 375 >125 >125 29 ± 14 >125 >125 >125 >125 >125 377 >125 >125104 >125 >125 >125 >125 >125 378 >125 >125 53.4 >125 >125 >125 >125 >125380 71 ± 18 106 ± 20 18 ± 7  94 ± 19 93 ± 23 69 ± 4 67 ± 12 68381 >125 >125 22 ± 8  >125 >125 >125 >125 >125

TABLE 5 BCL6 BCL6 dependent Kd independent GI₅₀ GI₅₀ Compound (μM) GI₅₀Toledo (μM) Ly7 (μM) SUDHL6 (μM) 2099 5.5 ± 0.7 75 65 30 ± 7

Accordingly, the invention provides, in various embodiments, a compoundof formula I

wherein a dashed line indicates that a double bond can be present orabsent; when a double bond is present, R³ is absent; R¹ is H,(C1-C6)alkyl, benzyl, 2-propenyl or 2-propynyl, or R¹ is a group offormula —CH₂CO₂R or of —CH₂C(═O)OCH(R)—Ar¹, wherein R is H or(C1-C6)alkyl, Ar¹ is phenyl substituted with 0, 1, or 2 independentlyselected substituents from the group consisting of (C1-C6)alkyl,(C1-C6)alkoxy, halo, and (C1-C6)haloalkyl; n=1, 2, or 3; R³ is H or OH;each of R⁴, R⁵, R⁶ and R⁷ is independently selected H, F, Cl, Br, or I;X is O or S; Y is OH or O(C1-C6)alkyl; or a pharmaceutically acceptablesalt thereof; provided that when R¹ is H, Y is OH, the double bondindicated by the dashed line is present and R³ is absent, and n=1, 2, or3, not all of R⁴, R⁵, R⁶ and R⁷ are H; and when R¹ is H, methyl, or2-propenyl, Y is OH, the double bond indicated by the dashed line ispresent and R³ is absent, n=1, 2 or 3, and R⁴, R⁵ and R⁷ are H, R⁶ isnot bromo.

The compound can be any of the compounds of Table 1 or Table 3, above,i.e., the compound can be any one of 1085, 1093, 1095, 1097, 1113, 1115,1117, 1165, 1167, 1169, 2001, 2003, 2005, 2021, 2023, 2025, 2027, 2031,2033, 2035, 2037, 2039, 2041, 2097, 2099, 2101, 3021, 3022, 3039, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, or 384, or apharmaceutically acceptable salt thereof.

The invention also provides, in various embodiments, a method ofdisrupting BCL6 BTB domain interactions with corepressors, in B-cells,comprising exposing the B cells to an effective concentration of acompound that blocks the lateral groove of BCL6.

The invention also provides, in various embodiments, a method ofinhibiting DLBCL tumor growth, or causing DLBCL tumor regression, orboth, in a mammal, comprising administering to the mammal an effectivedose of a compound that blocks the BTB lateral groove of BCL6.

The invention also provides, in various embodiments, a method ofinhibiting transcriptional repression induced by a complex of BCL6 withSMRT or other corepressor proteins in cancer cells, comprising exposingthe cancer cells to an effective concentration of a compound that blocksthe BTB lateral groove of BCL6.

The invention also provides, in various embodiments, a method oftreatment of a patient afflicted with cancer, comprising administeringto the patient an effective dose of a compound that blocks the BTBlateral groove of BCL6.

The expressions “effective amount” or “effective dose”, when used todescribe therapy to an individual suffering from a disorder, refers tothe quantity or concentration of a compound of the invention that iseffective to inhibit or otherwise act on BCL6 in the individual'stissues wherein BCL6 involved in the disorder, wherein such inhibitionor other action occurs to an extent sufficient to produce a beneficialtherapeutic effect.

“Treating” or “treatment” within the meaning herein refers to analleviation of symptoms associated with a disorder or disease, orinhibition of further progression or worsening of those symptoms, orprevention or prophylaxis of the disease or disorder, or curing thedisease or disorder. Similarly, as used herein, an “effective amount” ora “therapeutically effective amount” of a compound of the inventionrefers to an amount of the compound that alleviates, in whole or inpart, symptoms associated with the disorder or condition, or halts orslows further progression or worsening of those symptoms, or prevents,or provides prophylaxis for, the disorder or condition. In particular, a“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of compounds of the invention areoutweighed by the therapeutically beneficial effects.

In carrying out any one of these methods, the compound that blocks thelateral groove of BCL6 can be a compound of formula I, or can be any oneof the specific compounds of Table 1 or Table 3, i.e., can be any one of1085, 1093, 1095, 1097, 1113, 1115, 1117, 1165, 1167, 1169, 2001, 2003,2005, 2021, 2023, 2025, 2027, 2031, 2033, 2035, 2037, 2039, 2041, 2097,2099, 2101, 3021, 3022, 3039, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, or 384, or a pharmaceutically acceptable saltthereof.

SILCS analysis indicated the presence of HDCH binding pocket and, incombination with experimental data on 79-6 analogs, showed that both theindole fragment and the carboxylate end are required for the binding of79-6, while the rhodanine ring system is not essential.

Based on the SILCS data a new class of inhibitors for BCL6 were designedin which the chemical structure of 1085 was extended with an additionalhydrophobic group. A collection of 18 analogs was designed andsynthesized to 1) keep the overall planar configuration of the inhibitorand, 2) provide hydrophobic and hydrogen bonding functional groups thatwould interact with the HDCH pocket and, thus, further improve thepotency and specificity of the inhibitors. SILCS calculations indicatethat an additional aliphatic or aromatic system (green/purple mesh)added on NH group of the indole ring would increase potency.Accordingly, we have extended the indole fragment with hydrophobicgroups such as alkyl, benzyl, phenyl acetate, and benzyl acetate orbenzyl acetamide.

Additional compounds were next designed and synthesized with therhodanine ring removed from the scaffold. These novel inhibitorstargeting BCL6 are capable of binding simultaneously to the aromaticpocket and to the HDCH site but omit the rhodanine and carboxylic acidmoieties. Omission of the carboxylic acid moiety, though essential for79-6 and 1085 binding, was performed to determine if addition ofmoieties targeting the HDCH sites would compensate for the loss of thatgroup.

In various embodiments, the invention provides a compound of formula II,as described below, and in various embodiments provides a compound offormula II for carrying out any of the above-described methods ofblocking the BTB lateral groove of BCL6 with an effective amount orconcentration of the compound of formula II, including a compound of the2071/3033 series.

The invention provides a compound of the 2071/3033 series of formula II,or the use of a compound of Table 7, below, for carrying out any of themethods of blocking the BTB lateral groove of BCL6.

Accordingly, the invention provides a compound of formula II

wherein Z is O, CH₂, CCl₂, or C(═O); R¹ is a group of formula —CH₂CO₂R,—CH₂C(═O)OCH(R)—Ar¹ or —CH₂C(═O)N(R)CH(R)—Ar¹, wherein each R isindependently H or (C1-C6)alkyl, Ar¹ is phenyl or heteroaryl substitutedwith 0, 1, or 2 independently selected substituents from the groupconsisting of (C1-C6)alkyl, (C1-C6)alkoxy, halo, NR₂,N(R)C(═O)O(C1-C6)alkyl, and (C1-C6)haloalkyl; each of R⁴, R⁵, R⁶ and R⁷is independently selected H, F, Cl, Br, or I; or a pharmaceuticallyacceptable salt thereof.

For instance, the compound used for carrying out a method of theinvention can be any of compounds 2071, 2073, 2175, 3031, 3033, 3045,3047, 3049, 3051, 3053, or 3059, or a pharmaceutically acceptable saltthereof.

For ease of reference, it is noted that for formula II above, when Z isC═O, the compound is termed the 2071 series, and when Z is O, thecompound is termed as belonging to the 3033 series. FIG. 4 shows a graphof bioactivities of compound of the 2071 series analogous to the resultsshown in FIG. 1 for the 1085 series. FIGS. 5,6, and 7, show graphs ofbioactivities of compounds of the 3033 series and some additionalcompounds of the 1085 and 2071 series, analogous to the results shown inFIG. 1 for the 1085 series.

Accordingly, the invention provides, in various embodiments, a compoundof formula II for carrying out a method of disrupting BCL6 BTB domaininteractions with corepressors, in B-cells; a method of inhibiting DLBCLtumor growth, or causing DLBCL tumor regression, or both, in a mammal; amethod of inhibiting transcriptional repression induced by a complex ofBCL6 with SMRT or other corepressor proteins in cancer cells; and, amethod of treatment of a patient afflicted with cancer; all comprisingadministering to the patient an effective dose of a compound of formulaII that blocks the BTB lateral groove of BCL6.

All single enantiomer, diastereomeric, and racemic forms of a structureare intended, unless a particular stereochemistry or isomeric form isspecifically indicated. In several instances though an individualstereoisomer is described among specifically claimed compounds, thestereochemical designation does not imply that alternate isomeric formsare less preferred, undesired, or not claimed. Compounds used in thepresent invention can include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions, at anydegree of enrichment. Both racemic and diastereomeric mixtures, as wellas the individual optical isomers can be isolated or synthesized so asto be substantially free of their enantiomeric or diastereomericpartners, and these are all within the scope of the invention.

As used herein, the terms “stable compound” and “stable structure” aremeant to indicate a compound that is sufficiently robust to surviveisolation to a useful degree of purity from a reaction mixture, andformulation into an efficacious therapeutic agent. Only stable compoundsare contemplated herein.

Standard abbreviations for chemical groups such as are well known in theart are used; e.g., Me=methyl, Et=ethyl, i-Pr=isopropyl, Bu=butyl,t-Bu=tert-butyl, Ph=phenyl, Bn=benzyl, Ac=acetyl, Bz=benzoyl, and thelike.

The compounds described herein can be prepared in a number of ways basedon the teachings contained in the Synthetic Schemes and Examplesprovided herein and synthetic procedures known in the art to theordinary practitioner. In the description of the synthetic methodsdescribed below, it is to be understood that all proposed reactionconditions, including choice of solvent, reaction atmosphere, reactiontemperature, duration of the experiment and workup procedures, can bechosen to be the conditions standard for that reaction, unless otherwiseindicated. It is understood by one skilled in the art of organicsynthesis that the functionality present on various portions of themolecule should be compatible with the reagents and reactions proposed.Substituents not compatible with the reaction conditions will beapparent to one skilled in the art, and alternate methods are thereforeindicated. The starting materials for the examples are eithercommercially available or are readily prepared by standard methods fromknown materials. All commercially available chemicals were obtained fromAldrich, Alfa Aesare, Wako, Acros, Fisher, Fluka, Maybridge or the likeand were used without further purification, except where noted. Drysolvents are obtained, for example, by passing these through activatedalumina columns.

The present invention further embraces isolated compounds of theinvention or for practice of a method of the invention. The expression“isolated compound” refers to a preparation of a compound of theinvention, or a mixture of compounds the invention, wherein the isolatedcompound has been separated from the reagents used, and/or byproductsformed, in the synthesis of the compound or compounds. “Isolated” doesnot mean that the preparation is technically pure (homogeneous), but itis sufficiently pure to compound in a form in which it can be usedtherapeutically. The compounds of the invention and intermediates may beisolated from their reaction mixtures and purified by standardtechniques such as filtration, liquid-liquid extraction, solid phaseextraction, distillation, recrystallization or chromatography, includingflash column chromatography, or HPLC.

Isolated optical isomers may be purified from racemic mixtures bywell-known chiral separation techniques. According to one such method, aracemic mixture of a compound of the invention, or a chiral intermediatethereof, is separated into 99% wt. % pure optical isomers by HPLC usinga suitable chiral column, such as a member of the series of DAICEL®CHIRALPAK® family of columns (Daicel Chemical Industries, Ltd., Tokyo,Japan). The column is operated according to the manufacturer'sinstructions.

The compounds of the invention can be administered to a mammal,especially a human in need of such treatment, prevention, elimination,alleviation or amelioration of a malcondition. Such mammals include alsoanimals, both domestic animals, e.g. household pets, farm animals, andnon-domestic animals such as wildlife.

The compounds of the invention are effective over a wide dosage range.For example, in the treatment of adult humans, dosages from about 0.05to about 5000 mg, preferably from about 1 to about 2000 mg, and morepreferably between about 2 and about 2000 mg per day can be used. Atypical dosage is about 10 mg to about 1000 mg per day. In choosing aregimen for patients it can frequently be necessary to begin with ahigher dosage and when the condition is under control to reduce thedosage. The exact dosage will depend upon the activity of the compound,mode of administration, on the therapy desired, form in whichadministered, the subject to be treated and the body weight of thesubject to be treated, and the preference and experience of thephysician or veterinarian in charge.

Generally, the compounds of the invention are dispensed in unit dosageform including from about 0.05 mg to about 1000 mg of active ingredienttogether with a pharmaceutically acceptable carrier per unit dosage.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermaladministration include from about 125 μg to about 1250 mg, preferablyfrom about 250 μg to about 500 mg, and more preferably from about 2.5 mgto about 250 mg, of the compounds admixed with a pharmaceuticallyacceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such astwice or thrice daily. Alternatively dosage forms can be administeredless frequently than daily, such as every other day, or weekly, if foundto be advisable by a prescribing physician.

It is within ordinary skill to evaluate any compound disclosed andclaimed herein for effectiveness in inhibition of BCL6 binding tocorepressors and in the various cellular assays using the proceduresdescribed above or found in the scientific literature. Accordingly, theperson of ordinary skill can prepare and evaluate any of the claimedcompounds without undue experimentation.

Any compound found to be an effective inhibitor of BCL6 binding tocorepressors can likewise be tested in animal models and in humanclinical studies using the skill and experience of the investigator toguide the selection of dosages and treatment regimens.

TABLE 6 Compounds of the 2071/3033 series and others

2073

2071

2175

3031

3033

3045

3047

3049

3051

3053

3059

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

3033

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

2077

385

386

387

388

389

390

391

392

394

395

396

397

399

400

401

402

403

Tables 7 and 8 show the effects of compounds of the 2071 series on BCL6dependent and independent cell lines, and Tables 9 and 10 provideanalogous data for compounds of the 3033 series.

TABLE 7 BCL6 BCL6 dependent independent GI₅₀ GI₅₀ Compound Kd (μM) GI₅₀Toledo (μM) Ly7 (μM) SUDHL6 (μM) 2071 NB >125 53 35 2073 NB 40 30 30

TABLE 8 Cell line Bcl-6 independent Bcl-6 dependent Karpas WSU- Ly3 Ly7SUDH6 422 Toledo DLCL2 Compound GI₅₀ (μM) 2071 >40 26 ± 1 10 ±1 >40 >40 >40 2073 >40 36 ± 7 17 ± 1 >40 >40 >40 2075 >40  4.4 ± 0.7 14± 8 >40 >40 >40 2097 >30 >30 >30 >30 >30 >302099 >30 >30 >30 >30 >30 >30 2101 >30 >30 >30 >30 >30 >30

TABLE 9 Bcl6 dependent Bcl6 independent GI 50 (μM) Ly3 Ly1 Ly10 SUDHL6Ly7 Toledo K422 WSUDLCL2 Ly4 Ly1B50 355 54 37 62 1 45 35 38 52 81 55 35779 81 60 1 25 51 58 100 >125 56 358 >125 >125 >125 2 2069 >125 >125 >125 >125 361 56 24 166 7 26 39 20 41 98 103

TABLE 10 BCL6 independent BCL6 dependent GI₅₀ GI₅₀ SUDHL6 Compound Kd(μM) Toledo (μM) GI₅₀ Ly7 (μM) (μM) 333 NB >125 >125 >125 334NB >125 >125 >125 339 NB >125 >125 >125 341 NB >125 >125 >125 355 NB 45± 7  43 ± 3   42 ± 10 357 NB 50 ± 1  30 ± 10 22 ± 3 358 NB 82 ± 20 40 ±10 27 ± 4 367 324 ± 34 53 ± 10 22 ± 4  15 ± 7

Ligand design next focused on linking a bicyclic aromatic system thatbinds in the aromatic site with a moiety that interacts irreversiblywith the HDCH site. Compounds were designed that include reactivemoieties targeting Cys53 on the HDCH site, with the goal of developingof BCL6-specific irreversible inhibitors (compounds 3065, 3061, 3079,3063, 3055 and 3077, termed the 3055 series). Examples of suchirreversible inhibitors are shown in Table 11. Biodata are provided ingraphical form in FIG. 9.

TABLE 11 Compounds of the 3055 series: irreversible inhibitors

3066

3061

3079

3063

3055

3077

FIG. 10 depicts structural information and computational data for acompound of Scaffold 2, designed to target both the aromatic and arenesites, the compounds of Scaffold 2 being of formula (V)

wherein the ring labeled A is a substituted or unsubstituted aryl orheteroaryl ring, R is H or is (CH₂)_(m)CO₂H; m=1, 2, or 3; each R¹ isindependently selected halo, (C1-C6)alkyl, (C1-C6)alkoxy, nitro, ortrifluoromethyl; n=0, 1, 2, or 3; or a pharmaceutically acceptable saltthereof.

Table 12 presents exemplary structures and Table 13 present biodata forselected representatives of compounds of Scaffold 2. Representativestructures are shown in FIG. 11(A) and biodata are provided in FIGS.11(B) and 11(C).

TABLE 12

1055

1057

1059

1061

1063

1065

1067

1069

1071

1073

TABLE 13 BCL6 independent BCL6 dependent GI₅₀ Toledo GI₅₀ SUDHL6Compound Kd (μM) (μM) GI₅₀ Ly7 (μM) (μM) 393 174 ± 25 >125 >125 >1251057  87 ± 12 >125 >125 >125 1059 NB >125 >125 >125 1061 180 ± 36 >12560 40 ± 10 1063 315 ± 24 >125 >125 >125

Compounds of formula V can be prepared according to synthetic scheme 3,below.

FIG. 12 depicts structural information and computational data for acompound of Scaffold 3 of formula VI

wherein the ring labeled A is a substituted or unsubstituted aryl orheteroaryl ring, each R¹ is independently selected halo, (C1-C6)alkyl,(C1-C6)alkoxy, nitro, or trifluoromethyl; n=1, 2, or 3; p=0, 1, 2, or 3;L is a linker comprising an alkyl chain optionally comprising one ormore ether oxygen atom, ester group, or amide group; or apharmaceutically acceptable salt thereof.

Compounds of formula VI can be prepared according to Synthetic Scheme 4,below. A “linker”, as the term is used herein, refers to a bifunctionalchain bonded at each end to the ring labeled A and to the oxindolenitrogen atom, respectively. A linker can be an alkyl chain, optionallycomprising ether oxygens atoms, ester groups, amide groups, and thelike.

FIG. 13 depicts structural information and computational data for acompound of Scaffold 4 of formula VII

wherein the ring labeled A is a substituted or unsubstituted aryl orheteroaryl ring, each R¹ is independently selected halo, (C1-C6)alkyl,(C1-C6)alkoxy, nitro, or trifluoromethyl; n=1, 2, or 3; p=0, 1, 2, or 3;X¹ is O or NR, X² is O or H₂; R is H or (C1-C6)alkyl; or apharmaceutically acceptable salt thereof.

Compounds of formula VII can be prepared according to Synthetic Scheme5, below.

FIG. 14 depicts structural information and computational data for acompound of Scaffold 5, structure VIII,

wherein X¹ is O or S; R is a group of formula —CH₂(CH₂)_(n)CO2H or H,provided one and only one R is H; n=0, 1, 2, or 3; X² is halo,(C1-C6)alkyl, (C1-C6)alkoxy, nitro, or trifluoromethyl; or apharmaceutically acceptable salt thereof; the compounds of which formulacan be prepared according to Synthetic Scheme 6, below. Exemplarycompounds of formula VIII include

FIG. 15 presents structural information and computational data for acompound of Scaffold 6, structure 6. Compounds can be prepared accordingto Synthetic Scheme 7.

FIG. 16 presents structural information and computational data for acompound of Scaffold 7. Compounds can be prepared according to SyntheticScheme 8.

FIG. 17 present structural information and computational data for acompound of Scaffold 8. Compounds can be prepared according to SyntheticScheme 9.

FIG. 18 presents structural information and computational data for acompound of Scaffold 9, structure X,

wherein n=0, 1, 2, or 3, and R is (C1-C6)alkyl, compounds of which typecan be prepared according to the Synthetic Scheme 10, below. FIG. 19presents biodata for an exemplary compound of Scaffold 9.

FIG. 20 presents structural information and computational data for acompound of Scaffold 10. The exemplary compound of structure 34 wasfound to be inactive. A synthetic route is also shown in FIG. 20.

FIG. 21 presents structural information and computational data for acompound of Scaffold 11, formula XI

wherein the ring labeled A is a substituted or unsubstituted aryl orheteroaryl; R is (C1-C6)alkyl, each of n1 and n2 are independently 1, 2,or 3; or a pharmaceutically acceptable salt thereof Compounds ofScaffold 11 can be prepared according to Synthetic Scheme 11.

FIG. 21 presents biodata for a compound of Scaffold 11.

FIGS. 23(A), (B), and (C) show compounds of Scaffold 12.

wherein Ar¹ is a substituted or unsubstituted aryl or heteroaryl, R² ishalo, (C1-C6)alkyl, (C1-C6)alkoxy, nitro, or trifluoromethyl; n=0, 1, 2,or 3; or a pharmaceutically acceptable salt thereof. Compounds ofScaffold 12 of formula (12) can be prepared by condensation of ahydrazide and an aldehyde, as shown in Synthetic Scheme 12.

FIGS. 25-28 present biodata for compounds of Scaffold 12.

The invention further provides a method of treatment of a patientafflicted with cancer, comprising administering to the patient aneffective dose of a compound that blocks the BTB lateral groove of BCL6,such as compounds of any of the Scaffolds or Examples provided herein,wherein in addition to administration of the compound that blocks theBTB lateral groove of BCL6, an effective amount of a second anticanceragent is administered to the patient.

For instance, the second anticancer agent is doxorubicin, vincristine,dexamethasone, mechloretamine, or comprises a combination ofcyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP).Evaluation of the BCL6 groove binding peptide BPI discussed above, inconjunction with doxorubicin, vincristine, dexamethosone,mechloretamine, and the CHOP combination, in cell lines OCI-LY10,OCI-LY7, OCI-LY1, OCI-LY3, Farage, SU-DHL4, and SU-DHL6, showed at leastadditive and in some cases synergistic efficacies in all combinationsexcept vincristine in OCI-LY10 and dexamethasone in OCI-LY1, where theeffect was less than additive.

DOCUMENTS CITED

-   “A small-molecule inhibitor of BCL6 kills DLBCL cells in vitro and    in vivo”, Cerchietti L C, Ghetu A F, Zhu X, Da Silva G F, Zhong S,    Matthews M, Bunting K L, Polo J M, Fares C, Arrowsmith C H, Yang S    N, Garcia M, Coop A, MacKerell A D Jr., Prive G G, Melnick A, Cancer    Cell, 2010 Apr. 13 17(4); 400-411.-   “A peptidomimetic inhibitor of BCL6 with potent antilymphoma effects    in vitro and in vivo”, Cerchietti L C, Yang S N, Shaknovic R, Hatzi    K, Polo J M, Chadburn A, Dowdy S F, Melnick A, Blood, 20098 Apr. 9;    113(5); 3397-3405.-   “Sequential transcription factor targeting for diffuse large B-cell    lymphomas”, Cerchietti L C, Polo J M, Da Silva G F, Farinha P,    Shaknovich R, Gascoyne R D, Dowdy S F, Melnick A., Cancer Res. 2008    May 1; 68(9); 3361-3369.-   “Anticancer therapy SMRT-ens up: targeting the BCL6-SMRT interaction    in B cell lymphoma”, Compton L A, Hiebert S W, Cancer Cell, 2010    Apr. 13; 17(4); 315-316. doi″10.1016/j.ccr.2010.03.012.

Examples

Compounds used in practice of methods of the invention can be preparedby the person of ordinary skill based on the synthetic schemes providedherein in conjunction with ordinary knowledge.

Synthesis and Characterization of New Compounds

General Method A: Knoevenagel Condensation.

To a mixture of chloroisatin (1.0 mmol),3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (205 mg, 1.0 mmol) andNaOAc (820 mg, 10.0 mmol) was added acetic acid (5.0 mL). The reactionwas allowed to stir at 105° C. for 30 min-12 h, then cooled to roomtemperature. To the reaction was added water (15 mL). The resultingmixture was sonicated to give an orange-red slurry. After filtration,the solid was washed with water (75 mL) and dried under high vacuum toyield the corresponding product as a red fine powder (71-92%):

(Z)-3-(5-(5-Chloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1085)

This compound was synthesized using general method A (89%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.6 Hz, 2H), 4.20-4.30 (t, J=8.0 Hz,2H), 6.90-7.00 (d, J=8.0 Hz, 1H), 7.40-7.50 (dd, J=2.0, 8.4 Hz, 1H),8.80-8.81 (d, J=2.0 Hz, 1H), 11.41 (s, 1H), 12.40-12.70 (br s, 1H); ¹³CNMR (100 MHz, DMSO-d₆) δ 30.8, 112.2, 121.0, 123.8, 126.0, 127.0, 132.3,132.8, 143.3, 166.6, 167.7, 171.7, 197.1; LC-TOF (M+H⁺) calcd forC₁₄H₁₀ClN₂O₄S₂ 369. found 369.

(Z)-3-(5-(5,7-Dichloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1093)

This compound was synthesized using general method A (80%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.6 Hz, 2H), 4.25-4.35 (t, J=8.0 Hz,2H), 7.10-7.15 (d, J=1.6 Hz, 1H), 8.77-8.80 (d, J=1.6 Hz, 1H), 11.86 (s,1H), 12.40-12.70 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 30.8, 115.6,122.2, 123.0, 125.6, 126.3, 131.3, 134.9, 140.9, 166.6, 167.8, 171.7,196.7; LC-TOF (M+H⁺) calcd for C₁₄H₉Cl₂N₂O₄S₂ 403. found 403.

(Z)-3-(5-(5-Chloro-7-methyl-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1095)

This compound was synthesized using general method A (83%): ¹H NMR (400MHz, DMSO-d₆) δ 2.23, (s, 3H), 2.60-2.70 (t, J=7.6 Hz, 2H), 4.20-4.30(t, J=8.0 Hz, 2H), 7.35 (s, 1H), 8.67 (s, 1H), 11.43 (s, 1H),12.40-12.70 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 16.6, 31.2, 121.0,122.6, 124.8, 126.3, 133.7, 142.5, 167.0, 168.6, 172.2, 197.6; LC-TOF(M+H⁺) calcd for C₁₅H₁₂ClN₂O₄S₂ 383. found 383.

(Z)-3-(5-(5-Methyl-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1097)

This compound was synthesized using general method A (90%): ¹H NMR (400MHz, DMSO-d₆) δ 2.32, (s, 3H), 2.60-2.70 (t, J=7.6 Hz, 2H), 4.20-4.30(t, J=8.0 Hz, 2H), 6.80-6.90 (d, J=8.0 Hz, 1H), 7.20-7.25 (d, J=8.0 Hz,1H), 8.64 (s, 1H), 11.16 (s, 1H), 12.40-12.70 (br s, 1H); ¹³C NMR (100MHz, DMSO-d₆) δ 20.8, 30.7, 110.4, 119.8, 125.4, 128.1, 130.3, 130.8,133.6, 142.4, 166.4, 167.9, 171.7, 197.4; LC-TOF (M+H⁺) calcd forC₁₅H₁₃N₂O₄S₂ 349. found 349.

(Z)-3-(5-(5-Bromo-7-methoxy-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1113)

This compound was synthesized using general method A (85%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.6 Hz, 2H), 4.20-4.26 (t, J=8.0 Hz,2H), 4.32 (s, 3H), 7.36 (s, 1H), 8.60 (s, 1H), 11.51 (s, 1H),12.30-12.70 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 30.8, 111.6, 114.1,114.4, 119.3, 119.6, 124.5, 132.7, 141.1, 156.4, 158.7, 166.7, 168.0,171.8, 197.2; LC-TOF (M+H⁺) calcd for C₁₅H₁₂BrN₂O₅S₂ 443. found 443.

(Z)-3-(5-(5-Fluoro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1115)

This compound was synthesized using general method A (86%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.6 Hz, 2H), 4.20-4.26 (t, J=8.0 Hz,2H), 6.90-7.00 (dd, J=4.0, 8.0 Hz, 1H), 7.20-7.30 (m, 1H), 8.50-8.60 (d,J=8.0 Hz, 1H), 11.31 (s, 1H), 12.30-12.70 (br s, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 30.8, 111.6, 114.1, 114.4, 119.3, 119.6, 124.5, 132.7, 141.1,156.4, 158.7, 166.7, 168.0, 171.8, 197.2; LC-TOF (M+H⁺) calcd forC₁₄H₁₀FN₂O₄S₂ 353. found 353.

(Z)-3-(5-(7-Fluoro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1117)

This compound was synthesized using general method A (92%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.2 Hz, 2H), 4.20-4.26 (t, J=8.0 Hz,2H), 7.10-7.20 (m, 1H), 7.36-7.42 (dd, J=8.8, 9.2 Hz, 1H), 11.83 (s,1H), 12.54 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 30.8, 119.5, 119.7,122.5, 122.6, 122.8, 122.9, 123.9, 124.2, 124.3, 131.5, 131.7, 132.9,145.5, 147.9, 166.5, 167.9, 171.8, 197.2; LC-TOF (M+H⁺) calcd forC₁₄H₁₀FN₂O₄S₂ 353. found 353.

(Z)-3-(5-(5-Bromo-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1165)

This compound was synthesized using general method A (85%): ¹H NMR (400MHz, DMSO-d₆) δ 2.46-2.57 (t, J=7.6 Hz, 2H), 4.20-4.26 (t, J=7.6 Hz,2H), 6.80-6.90 (d, J=7.6 Hz, 1H), 7.52-7.57 (d, J=8.0 Hz, 1H), 8.93 (s,1H), 11.39 (s, 1H), 12.30-12.70 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ30.8, 112.7, 113.7, 121.6, 123.7, 129.8, 135.1, 143.7, 166.7, 167.7,171.7, 197.1; LC-TOF (M+H⁺) calcd for C₁₄H₁₀BrN₂O₄S₂ 413. found 413.

(Z)-3-(5-(7-Chloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1167)

This compound was synthesized using general method A (90%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=8.0 Hz, 2H), 4.20-4.30 (t, J=7.6 Hz,2H), 7.10-7.15 (t, J=8.0 Hz, 1H), 7.50-7.55 (d, J=7.6 Hz, 1H), 8.70-8.80(d, J=8.0 Hz, 1H), 11.71 (s, 1H), 12.40-12.80 (br s, 1H); ¹³C NMR (100MHz, DMSO-d₆) δ 30.8, 114.9, 121.5, 123.3, 124.3, 126.3, 132.4, 133.0,141.9, 166.5, 168.0, 171.8, 197.1; LC-TOF (M+H⁺) calcd forC₁₄H₁₀ClN₂O₄S₂ 369. found 369.

(Z)-3-(5-(5-Nitro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-1169)

This compound was synthesized using general method A (86%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (m, 2H), 4.20-4.30 (m, 2H), 7.00-7.10 (d,J=8.4 Hz, 1H), 8.20-8.30 (dd, J=1.6, 8.4 Hz, 1H), 9.50-9.60 (d, J=1.6Hz, 1H), 11.92 (s, 1H), 13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 31.1, 111.4, 120.2, 123.2, 129.0, 135.0, 142.7, 150.1, 167.1,168.8, 172.1, 197.0; LC-TOF (M+H⁺) calcd for C₁₄H₁₀N₃O₆S₂ 380. found380.

(Z)-3-(4-Oxo-5-(2-oxoindolin-3-ylidene)-2-thioxothiazolidin-3-yl)propanoicacid (FX-2001)

This compound was synthesized using general method A (75%): ¹H NMR (400MHz, DMSO-d₆) δ 1.12-1.16 (t, J=4.0 Hz, 3H), 2.60-2.75 (t, J=8.0 Hz,2H), 4.00-4.10 (m, 2H), 4.20-4.30 (m, 2H), 6.90-6.95 (d, J=8.0 Hz, 1H),7.40-7.50 (dd, J=2.0, 8.0 Hz, 1H), 8.75-8.80 (d, J=2.0 Hz, 1H), 11.37(s, 1H), 13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.4,30.3, 59.9, 111.7, 120.5, 123.4, 125.5, 131.8, 132.2, 142.9, 166.1,167.2, 169.6, 196.6; LC-TOF (M+H⁺) calcd for C₁₄H₁₁N₂O₄S₂ 335. found335.

(Z)-Ethyl3-(5-(5-chloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoate(FX-2003)

This compound was synthesized using general method A (74%): ¹H NMR (400MHz, DMSO-d₆) δ 1.12-1.16 (t, J=4.0 Hz, 3H), 2.60-2.75 (t, J=8.0 Hz,2H), 4.00-4.10 (m, 2H), 4.20-4.30 (m, 2H), 6.90-6.95 (d, J=8.0 Hz, 1H),7.40-7.50 (dd, J=2.0, 8.0 Hz, 1H), 8.75-8.80 (d, J=2.0 Hz, 1H), 11.37(s, 1H), 13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.4,30.3, 59.9, 111.7, 120.5, 123.4, 125.5, 131.8, 132.2, 142.9, 166.1,167.2, 169.6, 196.6; LC-TOF (M+H⁺) calcd for C₁₆H₁₄ClN₂O₄S₂ 398. found398.

(Z)-3-(5-(5-Iodo-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanoicacid (FX-2005)

This compound was synthesized using general method A (88%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=8.0 Hz, 2H), 4.20-4.30 (m, 2H),6.80-6.85 (d, J=8.0 Hz, 1H), 7.70-7.75 (d, J=8.0 Hz, 1H), 9.12 (s, 1H),11.40 (s, 1H), 13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ30.8, 85.2, 113.1, 122.0, 123.6, 132.6, 135.5, 140.9, 144.1, 166.7,167.5, 171.8, 197.2; LC-TOF (M+H⁺) calcd for C₁₀H₁₆N₂O₄ 229. found 229.

(Z)-2-(5-(5-Chloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2031)

This compound was synthesized using general method A (85%): ¹H NMR (400MHz, DMSO-d₆) δ 4.76 (s, 2H), 6.90-6.96 (d, J=8.8 Hz, 1H), 7.40-7.45(dd, J=1.6, 8.0 Hz, 1H), 8.70 (s, 1H), 11.39 (s, 1H), 13.00-14.00 (br s,1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 45.2, 112.7, 121.4, 125.5, 126.5,127.4, 131.7, 133.1, 144.1, 166.8, 167.6, 167.6, 168.0, 197.4; LC-TOF(M+H⁺) calcd for C₁₃H₈ClN₂O₄S₂ 355. found 355.

(Z)-2-(5-(5-Fluoro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2033)

This compound was synthesized using general method A (85%): ¹H NMR (400MHz, DMSO-d₆) δ 4.76 (s, 2H), 6.90-7.00 (dd, J=4.0, 8.0 Hz, 1H),7.26-7.30 (m, 1H), 8.40-8.50 (dd, J=2.8, 10.0 Hz, 1H), 11.29 (s, 1H),13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 44.9, 1118, 114.3,114.5, 119.8, 120.1, 125.8, 131.2, 141.5, 156.5, 158.8, 166.5, 167.3,168.0, 197.2; LC-TOF (M+H⁺) calcd for C₁₃H₈FN₂O₄S₂ 339. found 339.

(Z)-2-(5-(7-Fluoro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2035)

This compound was synthesized using general method A (72%): ¹H NMR (400MHz, DMSO-d₆) δ 4.78 (s, 2H), 7.00-7.15 (m, 1H), 7.36-7.41 (m, 1H),8.50-8.65 (d, J=7.6 Hz, 1H), 11.84 (s, 1H), 13.00-14.00 (br s, 1H); ¹³CNMR (100 MHz, DMSO-d₆) δ 45.1, 120.2, 120.4, 122.7, 123.2, 124.2, 125.7,131.6, 132.3, 145.8, 148.2, 166.4, 167.6, 168.0, 197.3; LC-TOF (M+H⁺)calcd for C₁₃H₈FN₂O₄S₂ 339. found 339.

(Z)-2-(5-(7-Chloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2037)

This compound was synthesized using general method A (75%): ¹H NMR (400MHz, DMSO-d₆) δ 4.78 (s, 2H), 7.08-7.12 (dd, J=8.0, 8.0 Hz, 1H),7.49-7.51 (d, J=8.0 Hz, 1H), 8.70-8.75 (d, J=8.0 Hz, 1H), 11.72 (s, 1H),13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 45.1, 115.3, 121.8,123.6, 125.8, 126.6, 131.7, 133.1, 142.6, 166.4, 167.5, 168.2, 197.3;LC-TOF (M+H⁺) calcd for C₁₃H₈ClN₂O₄S₂ 355. found 355.

(Z)-2-(5-(5,7-Dichloro-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2039).

This compound was synthesized using general method A (71%): ¹H NMR (400MHz, DMSO-d₆) δ 4.81 (s, 2H), 7.70 (s, 1H), 8.74 (s, 1H), 8.85 (s, 1H),11.90 (s, 1H), 13.00-14.00 (br s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ44.4, 115.7, 122.1, 124.3, 125.6, 131.6, 133.2, 141.3, 165.9, 166.3,166.9, 167.2, 167.7, 194.6, 196.7; LC-TOF (M+H⁺) calcd forC₁₃H₇Cl₂N₂O₄S₂ 389. found 389.

(Z)-2-(5-(5-Bromo-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)aceticacid (FX-2041)

This compound was synthesized using general method A (72%): ¹H NMR (400MHz, DMSO-d₆) δ 4.77 (s, 2H), 6.87-6.89 (d, J=8.0 Hz, 1H), 7.55-7.57 (d,J=7.6 Hz, 1H), 8.85 (s, 1H), 11.40 (s, 1H), 13.00-14.00 (br s, 1H); ¹³CNMR (100 MHz, DMSO-d₆) δ 45.2, 113.2, 114.2, 121.9, 125.3, 127.3, 130.2,131.8, 135.8, 144.5, 166.8, 167.7, 167.9, 197.4; LC-TOF (M+H⁺) calcd forC₁₃H₈BrN₂O₄S₂ 399. found 399.

Ethyl 2-(3-(4-oxo-2-thioxothiazolidin-3-yl)propanamido)acetate (FX-3019)

To a mixture of 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (205mg, 1.0 mmol), H₂N-Gly-OEt hydrochloride (140 mg, 1.0 mmol) and EDC (192mg, 1.0 mmol) in DMF (5.0 mL) was added triethylamine (140 μL, 1.0mmol). The reaction mixture was stirred at room temperature for 16 h,and then concentrated. The crude product was purified by flashchromatography (EtOAc/Hexanes 1:2-1:1) to give FX-3019 as a white solid(276 mg, 0.95 mmol, 95%): ¹H NMR (400 MHz, DMSO-d₆) δ 1.25-1.30 (t,J=6.8 Hz, 3H), 2.64-2.70 (t, J=7.6 Hz, 2H), 4.01 (s, 3H), 4.20-4.25 (dd,J=7.2, 14.0 Hz, 2H), 4.25-4.35 (t, J=8.0 Hz, 2H), 6.04 (s, 1H); ¹³C NMR(100 MHz, DMSO-d₆) δ 14.1, 32.7, 35.4, 40.6, 41.4, 61.7, 169.4, 169.8,173.6, 201.0; LC-TOF (M+H⁺) calcd for C₁₀H₁₅N₂O₄S₂ 291. found 291.

(Z)-ethyl2-(3-(5-(5-Bromo-2-oxoindolin-3-ylidene)-4-oxo-2-thioxothiazolidin-3-yl)propanamido)acetate (FX-3021)

This compound was synthesized using general method A (75%): ¹H NMR (400MHz, DMSO-d₆) δ 1.15-1.20 (t, J=7.2 Hz, 3H), 2.50-2.65 (t, J=7.6 Hz,2H), 3.76-3.80 (d, J=5.6 Hz, 2H), 4.04-4.10 (dd, J=6.8, 15.0 Hz, 2H),4.20-4.30 (m, 2H), 6.97-7.00 (d, J=7.6 Hz, 1H), 7.47-7.49 (d, J=7.6 Hz,1H), 8.50 (s, 1H), 8.83 (s, 1H), 11.41 (s, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 14.1, 31.7, 40.4, 40.7, 60.4, 112.2, 121.1, 123.7, 126.0,127.0, 132.2, 133.0, 143.3, 166.7, 167.8, 169.6, 169.7, 197.1; LC-TOF(M+H⁺) calcd for C₁₈H₁₇BrN₃O₅S₂ 498. found 498.

(Z)-3-(5-(5-Chloro-2-oxoindolin-3-ylidene)-2,4-dioxothiazolidin-3-yl)propanoicacid (FX-3039)

This compound was synthesized using general method A (71%): ¹H NMR (400MHz, DMSO-d₆) δ 2.60-2.70 (t, J=7.6 Hz, 2H), 3.80-3.90 (t, J=7.6 Hz,2H), 6.90-7.00 (d, J=8.0 Hz, 1H), 7.40-7.50 (dd, J=2.4, 8.0 Hz, 1H),8.79-8.80 (d, J=2.4 Hz, 1H), 11.37 (s, 1H), 12.42 (br s, 1H); ¹³C NMR(100 MHz, DMSO-d₆) δ 31.4, 37.0, 112.0, 121.1, 125.9, 127.2, 132.0,142.6, 165.4, 168.1, 169.5, 171.9; LC-TOF (M+H⁺) calcd for C₁₄H₁₀ClN₂O₅S353. found 353.

Compounds of the 2071/3033 series can be prepared according to thefollowing Scheme, in conjunction with ordinary skill and knowledge.

Cell Culture

293T cells were grown in Dulbecco's modified Eagles medium supplementedwith 10% fetal bovine serum (FBS) (Gemini Bio-Products, Woodland,Calif.) and 1% penicillin-streptomycin. DLBCL cell lines OCI-Ly1,OCI-Ly1-B50, OCI-Ly7, OCI-Ly10 and OCI-Ly19 were grown in 80% Iscove'smedium, 20% FBS and penicillin G/streptomycin. DLBCL cell lines Toledo,Farage, SU-DHL4, DOHH-2, SC-1, RCK8, TMD8, HBL-1, Ly3, Karpas 422 andSU-DHL6 were cultured in 90% RPMI medium, 10% FBS, 2 mM glutamine, 10 mMHepes and penicillin G/streptomycin. All cell lines were cultured at 37°C. in a humidified atmosphere of 5% CO2.

MicroScale Thermophoresis Measurements:

Recombinant BCL6-BTB was labeled using the RED-NHS Labeling kit(NanoTemper Technologies). The labeling reaction was performed accordingto the manufacturer's instructions in the supplied labeling bufferapplying a concentration of 20 μM protein (molar dye:protein ratio≈2:1)at RT for 30 min. Unreacted dye was removed with the supplied dyeremoval columns equilibrated with PBS buffer (PBS, 0.005% Tween-80). Thelabel:protein ratio was determined using photometry at 650 nm andBradford reagent. Thereby, a ratio of 0.8 was typically achieved. Thelabeled BCL6-BTB was adjusted to 400 nM with PBS (Thermo) buffersupplemented with 0.05% Tween-80 (Fisher scientific). SMRT, 79-6 and1085 were dissolved in PBS buffer supplemented with 0.05% Tween-80 and10% DMSO and a series of 16 1:1 dilutions were prepared in the identicalbuffer, producing ligand concentrations ranging from 19 pM to 625 μM.For thermophoresis, each ligand dilution was mixed with one volume oflabeled BCL6-BTB, which leads to a final concentration of fluorescentlylabeled BCL6-BTB of 200 nM and final ligand concentrations ranging from9 pM to 312 nM in a 5% DMSO final concentration. After 10 minincubation, approximately 4 μL of each solution was filled into MonolithNT Standard Treated Capillaries (NanoTemper Technologies GmbH).Thermophoresis was measured using a Monolith NT.115 instrument(NanoTemper Technologies GmbH) at an ambient temperature of 25° C. with5 s/30 s/5 s laser off/on/off times, respectively. Instrument parameterswere adjusted with 90% LED power and 40% MST power. Data of threeindependent experiments were analyzed (NT.Analysis software version1.5.41, NanoTemper Technologies) using the signal from Thermophoresis.

Luciferase Reporter Assays

For screening of the synthesized small molecules we transfected 5×10⁵293 T cells in a 6-well plate using polyethylenimine (PEI) with aluciferase reporter vector containing five binding sites for the yeastGAL4 DNA binding domain and a thymidine kinase (TK) promoter,(GAL4)5TK-Luc (Polo et al., 2004) and an internal control TK-renillareporter vector, pRL-TK (Promega) at a 10:1 ratio. Cells were alsotransfected with 250 ng of a plasmid expressing the GAL4 DNA bindingdomain (DBD) alone (pBXG1) or GAL4-DBD fused to the BCL6-BTB.Alternatively, cells were transfected with 1320 ng of plasmid containingthe Kaiso-BTB domain fused to GAL4-DBD, 500 ng HIC-BTB-GAL4-DBD (Polo etal., 2004), 500 ng PLZF-BTB-GAL4-DBD (Polo et al., 2004), or 500-1320 ngGAL4-DBD alone. Twenty-four hours after transfection cells wereharvested and redistributed to 96-well plates at a density of 20,000cells per well, respectively, after which cells were treated inquadruplicate with 50 or 100 μM concentrations of different compounds orDMSO for 24 hr. Cell lysates were examined for the abundance of fireflyluciferase relative to renilla luciferase (in counts per second) withthe Dual-Luciferase Reporter Assay kit (Promega, Madison, Wis.)according to the manufacturer's protocol and a Synergy4 plate reader(BioTek Instruments, Winooski, Vt.). The repressor activity of each BTBdomain was calculated as the relative fold change in repression comparedwith the GAL4 DBD plasmid control under the same treatment conditions.

All patents and publications referred to herein are incorporated byreference herein to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. A method of disrupting BCL6 BTB domaininteractions with corepressors, in B-cells, comprising exposing the Bcells to an effective concentration of a compound that blocks thelateral groove of BCL6; wherein the compound that blocks the lateralgroove of BCL6 is a compound of formula (I)

wherein a dashed line indicates that a double bond can be present orabsent; when a double bond is present, R³ is absent; R¹ is H,(C1-C6)alkyl, benzyl, 2-propenyl or 2-propynyl, or R¹ is a group offormula —CH₂CO₂R or —CH₂C(═O)OCH(R)—Ar¹, wherein R is H or (C1-C6)alkyl,Ar¹ is phenyl substituted with 0, 1, or 2 independently selectedsubstituents from the group consisting of (C1-C6)alkyl, (C1-C6)alkoxy,halo, and (C1-C6)haloalkyl; n=2; R³ is H or OH; R⁴ and R⁶ are H, R⁵ isCl, and R⁷ is H or Cl; X is O or S; and Y is OH or O(C1-C6)alkyl; or apharmaceutically acceptable salt thereof.
 2. A method of inhibitingdiffuse large B-cell lymphoma (DLBCL) tumor growth, or causing DLBCLtumor regression, or both, in a mammal, comprising administering to themammal in need thereof an effective dose of a compound that blocks theBTB lateral groove of BCL6; wherein the compound that blocks the lateralgroove of BCL6 is a compound of formula (I)

wherein a dashed line indicates that a double bond can be present orabsent; when a double bond is present, R³ is absent; R¹ is H,(C1-C6)alkyl, benzyl, 2-propenyl or 2-propynyl, or R¹ is a group offormula —CH₂CO₂R or —CH₂C(═O)OCH(R)—Ar¹, wherein R is H or (C1-C6)alkyl,Ar¹ is phenyl substituted with 0, 1, or 2 independently selectedsubstituents from the group consisting of (C1-C6)alkyl, (C1-C6)alkoxy,halo, and (C1-C6)haloalkyl; n=2; R³ is H or OH; R⁴ and R⁶ are H, R⁵ isCl, and R⁷ is H or Cl; X is O or S; and Y is OH or O(C1-C6)alkyl; or apharmaceutically acceptable salt thereof.
 3. A method of inhibitingtranscriptional repression induced by a complex of BCL6 with SMRT orother corepressor proteins in cancer cells, comprising exposing thecancer cells to an effective concentration of a compound that blocks theBTB lateral groove of BCL6; wherein the compound that blocks the lateralgroove of BCL6 is a compound of formula (I)

wherein a dashed line indicates that a double bond can be present orabsent; when a double bond is present, R³ is absent; R¹ is H,(C1-C6)alkyl, benzyl, 2-propenyl or 2-propynyl, or R¹ is a group offormula —CH₂CO₂R or —CH₂C(═O)OCH(R)—Ar¹, wherein R is H or (C1-C6)alkyl,Ar¹ is phenyl substituted with 0, 1, or 2 independently selectedsubstituents from the group consisting of (C1-C6)alkyl, (C1-C6)alkoxy,halo, and (C1-C6)haloalkyl; n=2; R³ is H or OH; R⁴ and R⁶ are H, R⁵ isCl, and R⁷ is H or Cl; X is O or S; and Y is OH or O(C1-C6)alkyl; or apharmaceutically acceptable salt thereof.
 4. A method of alleviating thesymptoms of cancer in a patient in need thereof, comprisingadministering to the patient an effective dose of a compound that blocksthe BTB lateral groove of BCL6; wherein the compound that blocks thelateral groove of BCL6 is a compound of formula (I)

wherein a dashed line indicates that a double bond can be present orabsent; when a double bond is present, R³ is absent; R¹ is H,(C1-C6)alkyl, benzyl, 2-propenyl or 2-propynyl, or R¹ is a group offormula —CH₂CO₂R or —CH₂C(═O)OCH(R)—Ar¹, wherein R is H or (C1-C6)alkyl,Ar¹ is phenyl substituted with 0, 1, or 2 independently selectedsubstituents from the group consisting of (C1-C6)alkyl, (C1-C6)alkoxy,halo, and (C1-C6)haloalkyl; n=2; R³ is H or OH; R⁴ and R⁶ are H, R⁵ isCl, and R⁷ is H or Cl; X is O or S; and Y is OH or O(C1-C6)alkyl; or apharmaceutically acceptable salt thereof.
 5. The method of any one ofclaims 1-4, wherein the compound of formula (I) is any one of

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 4,wherein in addition to administration of the compound that blocks theBTB lateral groove of BCL6, an effective amount of a second anticanceragent is administered to the patient.
 7. The method of claim 6, whereinthe second anticancer agent is doxorubicin, vincristine, dexamethasone,or mechloretamine, or comprises a combination of cyclophosphamide,doxorubicin, vincristine, and prednisone (CHOP).